The production of various proteins and peptides with commercial value is the basis for the establishment and the rapid growth of the genetic engineering industry. This is facilitated by utilizing sophisticated recombinant DNA technologies rather than conventional synthetic routes used by the chemical industry, which are inapplicable. At the core of these technologies is the in vitro fermentation of microorganisms, typically a bacteria, containing the fight gene (in an appropriate expression vector) encoding the target protein. The fermentation is followed by "harvesting" the genetically engineered microorganisms and isolating the desired product through a series of separation and purification processes.
This method has some drawbacks. (a) Eukaryotic proteins expressed in microorganisms are often not properly folded; in particular, glycosylation and the proper conformation of complex proteins are not performed correctly. (b) Proteins expressed in large amounts often precipitate into insoluble aggregates (inclusion bodies) from which they can be recovered only by solubilization in denaturing agents. (c) Proteins which are secreted into the periplasmic space of the producer cell when produced in small amounts, often cannot be secreted there when produced in large amounts.
Compared with the above-mentioned bacterial systems, mammalian expression techniques have certain advantages, particularly for the expression of higher eukaryotic proteins. The expressed proteins are usually properly modified, and they almost always accumulate in the correct cellular compartment. So far, however, the mammalian expression techniques are practical for small and medium-scale work, but they are difficult and very expensive to perform on an industrial scale.
In eukaryotic organisms the synthesis of proteins is mediated by the ribosome scanning model. The (40S) ribosomal sub-unit (carrying Met-tRNA and various initiation factors) binds at the 5' end of the mRNA and then migrates in a 5'-3' direction, stopping at the first AUG codon in favorable context for initiating translation.
In eukaryotes, most cellular cytoplasmic mRNA's carry a 7-methylguanylate cap attached to their 5' end. The binding of the ribosomal sub-unit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap structure and the cap binding protein complex. This complex includes a eukaryotic translation-initiation factor eIF-4F (which contains the subunit p220).
Picornaviruses are mammalian and plant plus strand RNA viruses whose genomes serve as mRNAs. These viruses are capable of blocking translation of cellular mRNA without affecting translation of vital mRNA. The mechanism of this shut-off is related to inactivation of the eukaryotic translation initiation factor eIF-4F. Poliovirus (RNA+stranded picornavirus), induces the cleavage of the P220 (eIF-4F) and thus prevents the binding of capped mRNA to the 40S ribosome as part of the translation initiation process. The cleavage of the P220 component is done directly or indirectly by the poliovirus protease 2A, a translation inhibition factor (TIF). 2A is a protease, the mature form of which is generated by the cleavage of the precursor polyprotein of poliovirus at its N terminus by 2A itself and at its C terminus by another protease 3C [F. Toyoda et al., 1988, Cell 45, pp. 761-770]. The vital strategy therefore prevents cap dependent translation of cellular mRNA's, without affecting the cap independent translation of vital mRNA. A large segment of the viral 5' untranslated region (UTR), approximately 750 nucleotides in length, promotes "internal" entry of ribosomes without the need for 5' capping of the mRNA. Vital mRNA is therefore translated by a mechanism in which the ribosome binds directly to an internal site of the mRNA without any need for p220. Thus Picornaviruses cause a dramatic shut-off of host cell mRNA translation (within several hours after infection), while allowing the cap independent translation of vital mRNA.
It is known that few cellular mRNAs, such as the one encoding the immunoglobulin heavy chain binding protein (BiP) can be translated in poliovirus-infected cells at a time when cap dependent translation of all other host cell mRNA is inhibited. The 5' leader of BiP (UTR), like that of poliovirus, can directly confer internal ribosome binding to an mRNA in mammalian cells.
Zhou et. al. (Molecular and Cellular Biology, 1990, 10, 4529-4539) transiently co-transfect a cell line with a vector in which an internal ribosome entry site (IRES) precedes a reporter gene, and another vector in which an internal ribosome entry site (IRES) precedes the poliovirus 2A gene. These transfected cells express 2A for a limited period of time and allow only for transient expression of the reporter gene. Production of most proteins needed for cellular viability is arrested because translation of all capped mRNA is blocked, thus leading to cell death.
It is common knowledge that expression of 2A in cells can only be transient, due to cellular death. In a patent application of the same applicant herein (EP 585,983) expression systems permitting constitutive expression of the 2A protease in stable eukaryotic cell lines have been described. It is an object of the present invention to provide improved vectors which can be used in the expression systems of EP 585,983.
The following terms as used herein have the indicated meanings:
BiP--The human immunoglobuhn heavy chain-binding protein (also known as GRP 78). PA0 ETF--Eukaryotic translation Factor, e.g., the eIF-4F complex. PA0 Eukaryotic Promoter--A DNA fragment that precedes a gene and enhances its transcription in eukaryotic cells. PA0 Gene Product--a translation product, comprising--but not limited to--polypeptides and proteins. PA0 In vitro production--the production of one or more selected polypeptides in eukaryotic cell lines. PA0 IRES--Internal ribosome entry site. PA0 Polyfunctional cloning site--A DNA sequence that contains restriction sites for two or more restriction enzymes, and into which various genes can be cloned. PA0 Recombinant Nucleotide Vector--A vector including a recombinant nucleotide sequence. PA0 TIF--Eukaryotic translation inhibition factor, e.g. the poliovirus protease 2A. PA0 Transfection--The introduction of a vector containing a recombinant nucleotide sequence (DNA or an RNA) encoding one or more genes to be expressed into a host cell. PA0 UTR--an IRES containing the 5' untranslated region of picornavirus or of BiP. PA0 Vector--Any autonomously replicating or integrating agent, including but not limited to, plasmids, viruses, phages and the like. PA0 Vector system--The ensemble of two or more vectors which cooperate in the production of a gene product. PA0 Weak eukaryotic promoter--A eukaryotic promoter that precedes a TIF gene and allows its constitutive expression in stable eukaryotic cell lines.